GENE EXPRESSION IN SV40-TRANSFORMED HUMAN FIBROBLASTS

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Human diploid fibroblasts (HF) have a limited lifespan in culture, ending in a postmitotic state of senescence, which has been documented as a model for cellular aging. One approach toward an understanding of senescence is to examine mechanisms of overcoming it. The protein large T antigen (LT) encoded by the DNA virus SV40 transiently overcomes the growth arrest in senescent cells, extends the lifespan of cells beyond senescence, and, less frequently, permanently overcomes senescence generating immortal cells. We propose to investigate each of the three SV40-cell interactions and their interrelationships. Our working model is that viral gene expression (i.e. LT) promotes cellular proliferation and alterations in cellular genes which are responsible for overcoming senescence in a stable manner. We will particularly assess the role of cell death through apoptosis in limiting the growth of SV40-transformed HF in all three cell states. To facilitate this analysis, we have developed a series of matched pre-immortal and immortal SV40-transformed cell lines (SV/HF) including ones with a temperature-dependent growth behavior (SVtsA/HF and AR 5). Several questions will be addressed in this proposal in an effort to identify effector genes involved in overcoming senescence. We will determine whether the reported ability of LT to induce cellular DNA synthesis (DS) but not mitosis in senescent cells is actually due to a defect in DS, leading to a checkpoint arrest in G2. SV40 TL is able to induce mitosis upon transformation but its effect is temporary. We will evaluate the role of LT, p53, and apoptosis at the end of the extended lifespan of SVtsA/HF-C, termed crisis. In an effort to define novel functions responsible for crisis, we will analyze cDNA libraries generated from SVtsA/HF-C mRNAs overexpressed in crisis as compared to early in the period of extended lifespan. To assess the mitogen role of LT in extended lifespan and immortalization we will attempt to replace LT function by exploiting lines which are temperature-dependent for growth (AR5) into which c-myc has been introduced alone and together with other genes such as ras, and p53. Rare clones of AR5/cmyc which have overcome the temperature defect will also be characterized to determine the cellular basis for this effect. In conclusion, these studies should clarify the role of known factors and define unknown factors in overcoming senescence.